Methods for rapid multiplexed amplification of target nucleic acids

• US 8,425,861 B2
• Filed: 04/04/2008
• Issued: 04/23/2013
• Est. Priority Date: 04/04/2007
• Status: Active Grant

• Current Assignee: ANDE Corp.
• Original Assignee: NetBio, Inc.
• Inventors: Selden, Richard F., Tan, Eugene, Lam, Heung Chuan, Giese, Heidi Susanne, Kellogg, Gregory John, Wright, John A.
• Primary Examiner(s): Gordon, Brian R
• Assistant Examiner(s): Sasaki, Shogo
• Application Number:
  US12/080,746
• Publication Number:
  US 20090023603A1
• Time in Patent Office: 1,845 Days
• Field of Search: 435/92.1, 435/287.1, 435/288.4, 435/303.1, 422501-503, 506/43, 436/180
• US Class Current: 422/503
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2200/147 Employing temperature sensors
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: G01N 2021/6441 : with two or more labels
• CPC Class Codes: G01N 21/6402 : Atomic fluorescence; Laser induced fluorescence
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6486 : Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/06113 : Coherent sources; lasers
• CPC Class Codes: G01N 27/44726 : using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44782 : of a plurality of samples
• CPC Class Codes: G01N 27/44791 : Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A fast, multiplexed PCR system is described that can rapidly generate amplified nucleic acid products, for example, a full STR profile, from a target nucleic acid. Such systems include, for example, microfluidic biochips and a custom built thermal cycler, which are also described. The resulting STR profiles can satisfy forensic guidelines for signal strength, inter-loci peak height balance, heterozygous peak height ratio, incomplete non-template nucleotide addition, and stutter.

Claims

1. A thermal cycler system comprising:

a temperature control element (TCE) having a first surface;

a substrate comprising a sample chamber comprising a solution, said sample chamber positioned in thermal communication with said first surface, and a sensing chamber comprising a thermosensor, wherein the sensing chamber is fabricated to have the same material stack-up as the sample chamber, and configured and positioned in the substrate at an analogous position to that of the sample in the sample chamber so as to simulate conditions within said sample chamber; and

a controller configured to receive input from said thermosensor,

wherein said thermosensor provides feedback to heat or cool the TCE to set or maintain the solution at a desired temperature.

2. The thermal cycler system of claim 1, wherein said substrate is a biochip and wherein said sample chamber is a reaction chamber of said biochip.

3. An integrated biochip system comprising:

a biochip comprising

(i) a cover layer having a thickness of less than 500 μ

m; and

(ii) at least one reaction chamber formed therein and further comprising a solution, each of said reaction chambers in contact with said cover layer and further comprising a microfluidic inlet channel and a microfluidic outlet channel; and

(iii) at least one sensing chamber comprising a thermosensor, each of said sensing chambers fabricated to have the same material stack-up as the reaction chamber, and configured and positioned within the biochip at an analogous position to that of the solution in the reaction chamber so as to simulate the conditions within at least one reaction chamber; and

a thermal cycler system, comprising;

a temperature control element (TCE) having a first surface, wherein the first surface of said TCE is in thermal communication with the cover layer of the biochip, and;

a controller configured to receive input from said thermosensor, said thermosensor providing feedback to heat or cool the TCE to set or maintain a desired temperature of the solution contained within said at least one reaction chamber.